PROJECT SUMMARY The developmental program of the preimplantation embryo is modified by alterations in its microenvironment. The resultant change in development can have long-acting effects that extend into postnatal life. Inappropriate signaling between mother and embryo can lead to increased pregnancy loss and long-term changes in health. Research on developmental programming could lead to elimination of adverse outcomes associated with IVF and result in new approaches to improve human health and animal production generally. One characteristic of developmental programming during the preimplantation period is sexual dimorphism in the modification in adult phenotype. Sex differences in embryonic responses to changes in the microenvironment are likely mediated in part by sex-specific responses of the embryo to maternal regulatory signals. One molecule involved in this phenomenon is colony stimulating factor 2 (CSF2). In cattle, treatment of morula and blastocyst stage embryos with CSF2 changed embryonic characteristics at a later point in pregnancy (Day 15) in a sex-specific manner and, at least in female offspring, resulted in calves with increased growth rates in the first year of life. The long-term goal is to understand how the microenvironment of the embryo interacts with sex to shape the developmental program. The overall objectives of the current proposal are to 1) elucidate mechanisms by which CSF2 causes differential programming actions on male and female embryos and 2) characterize the consequences of actions of CSF2 on the preimplantation embryo in vitro and in vivo for subsequent embryonic and postnatal phenotype. It is hypothesized that sex-dependent responses to CSF2 depend upon differences between male and female embryos in remodeling of DNA methylation by CSF2 and that these changes in DNA methylation result in long-acting effects on the developmental program that have consequences for postnatal phenotype. There are three specific aims. For Aim 1, it will be tested whether differences between male and female preimplantation embryos in programming actions of CSF2 on the Day 15 embryo are dependent upon DNA methylation. For Aim 2, the importance of CSF2 for embryonic development in vivo will be evaluated by testing consequences of knocking out the CSF2 receptor for embryo elongation, gene expression and DNA methylation in male and female embryos. Aim 3 will focus on ascertaining whether exposure of the preimplantation female embryo to CSF2 programs development to alter postnatal phenotype in a manner that improves dairy cow productivity. Through these aims, we will elucidate importance of remodeling of the methylome for sex-dependent actions of CSF2 (Aims 1) and ascertain whether actions of CSF2 occur not only in vitro but also for embryos developing in utero (Aim 2). Finally, the experiment for Aim 3 represents the first attempt to enhance productivity of a food animal through use of a developmental programming molecule to manipulate development in the preimplantation period. .